Most conventional chemical or biochemical assays are based on “bulk” measurements. In such measurements, a collective behavior of a plurality of molecules within a certain volume of a sample solution is measured to determine the properties of the molecules. However, in many situations, bulk measurement approaches cannot be utilized, such as when sample volume is too small or the concentration of a target molecule is too low for a given technique's limit of sensitivity in detecting target molecules. In recent years, the detection of single molecules has become possible. Single-molecule detection offers much higher sensitivity and provides more detailed information than conventional bulk measurements. The development of single-molecule instrument sensitivity also promises new opportunities for high-sensitivity biological molecule detection and diagnosis, as described in Qiu, H., et al., “Fluorescence single-molecule counting assays for high-sensitivity detection of cytokines and chemokines”, CLINICAL CHEMISTRY 53(11):2010-2012 (2007).
A description of approaches for achieving single-molecule detection is provided in Moemer, W. E. and Fromm, D. P., “REVIEW ARTICLE: Methods of single-molecule fluorescence spectroscopy and microscopy”, REVIEW OF SCIENTIFIC INSTRUMENTS 74(8): 3597-3619 (2003) and in Walter, N. G., et al., “Do-it-yourself guide: how to use the modern single-molecule toolkit”, NATURE METHODS 5:475-489 (2008). These reviews also discuss methods and apparatuses known in the art that have been used or proposed for single-molecule detection. Applications of single-molecule detection include single-molecule DNA sequencing, single-molecule biomarker detection and miniaturized flow-cytometry-like detection.
Optimized systems and methods for single molecule detection have great potential for accelerating DNA sequencing technology. The Human Genome Project (HGP) spurred a great increase in DNA sequencing throughput. This increase, along with technical improvements, resulted in a corresponding drop in sequencing costs. While the first genome required 13 years and nearly three billion US dollars to completely sequence, it has been predicted that DNA sequencing technologies may ultimately become sufficiently affordable for personal genomics to be an integral component of routine clinical care (McGuire et al., SCIENCE 317:1687 (2007)). Personal genomes represent a paradigm shift in medical treatment for both patients and health care providers. By managing genetic risk factors for disease, health care providers can more readily practice preventative medicine and provide customized treatment. With large banks of completed genomes, drug design and administration can be more efficient, thereby accelerating the nascent field of pharmacogenomics. However, this acceleration will depend on the realization of robust, high-throughput, and low-cost DNA sequencing technologies.
To achieve single-molecule detection, an optical system must be able to selectively excite the molecule of interest in a complicated environment, and be able to avoid the interference from the background noise and detect the weak light emitted from that single molecule. One of the approaches for achieving single-molecule detection is locating the molecule of interest inside a confined space facilitating detection of light emitted from one molecule. U.S. Pat. No. 6,917,726 discloses a microscopy system incorporating a zero-mode waveguide (ZMW), which can facilitate the detection of single molecules by using nano-scale wells in which defined excitation light fields are created. Placement of molecules within these nanowells, and thus within these defined excitation light fields, greatly minimizes noise, thereby enhancing the detectability of light emitted from a single molecule. See also, for example, U.S. Pat. No. 6,917,726; U.S. Pat. No. 7,170,050; and U.S. Pat. No. 7,486,865. However, loading of the single-molecule sample into the excitation field requires attachment of the molecules to the bottom of the ZMW in the nanowells, which is difficult and inefficient (Eid et al., SCIENCE, 323:133-138 (2009)). Only wells containing one single-molecule sample are useful, while empty wells or wells containing multiple single-molecule samples are not.
Improved methods of sample loading with higher success rates are disclosed in U.S. patent publication no. 2010/0009872. These methods utilize a nano-scale particle to carry a single-molecule sample to the top opening of the nanowell and deliver the sample to the bottom of the well, where the sample may be exposed to the excitation field. However, the process involves many steps and chemical reactions to transfer the single-molecule sample from the nanoparticle carrier to the bottom of the well. Furthermore, the loaded wells are either impossible or difficult to reuse.
International Patent Publication Number WO 2009/017678 disclosed methods of single-molecule nucleic acid sequencing in which a single polymerase is immobilized on a surface to repeatedly sequence a circular nucleic acid template, thereby improving the accuracy of single-molecule sequencing. In this method for optical detection of single-molecule sequencing, immobilization of reactants directly on the surface confined reactions to within a zeptoliter volume. However, the difficulty of binding a single molecule of an enzyme or a nucleic acid onto the surface limits high-throughput use. Furthermore, a sequencing reaction in which a polymerase is immobilized directly on the surface will be terminated whenever the polymerase loses its activity, preventing completion of sequencing analysis at that site. In order to immobilize a molecule or enzyme in place, as set forth in WO 2009/017678, the region of surface must be well-defined and the chemical property of that region must be precisely controlled. This presents difficulties and added costs for device manufacturing.
Therefore, there is a need for improved systems and methods for detection of single-molecule objects.